In systems biology, and in the application of biomarker detection and biosensing, the separation and identification of many proteins, small molecules, and carbohydrates from a cell or from complex biological samples, is necessary. Often, one needs to profile the concentrations of many different biomarkers, cytokines and other signaling molecules contained in serum, to determine or diagnose the current progress of a disease. However, these biomarkers (typically smaller than 30 kD) are present at relatively low concentrations (pM˜nM), while majority proteins (albumin and globulins, typically larger than 40 kD) are present at much higher concentrations (μM˜mM), which critically limits the detection of the smaller biomarkers.
Pre-fractionation and separation could eliminate background molecules to enhance the detection ability of the signaling molecules, but none of the conventional separation techniques is appropriate for this task. Gel electrophoresis is routinely used for separating proteins based on size, but they are generally slow and hard to automate, and require bulky equipment. Capillary Electrophoresis (CE) with a liquid sieving matrix is currently the fastest size-based separation technique for protein, but polymeric sieving matrix can interfere with downstream separation and detection processes, which limits the automation of the entire sample preparation process. While microfluidic biomolecule separation systems hold much promise for miniaturizing and automating biomolecule analysis processes, most adopt the same gel sieve material in their separation, with all inherent limitations of the conventional techniques.
Micro/nanofluidic molecular sieving structures fabricated with semiconductor technology have been used to separate biomolecules as well, with much greater speed than their conventional counterparts, though to date the systems have only successfully been used for large biomolecule separation such as viral DNA based on size.
Thus, a high-throughput biomolecular sorter, which can be automated, and can be readily incorporated into downstream analysis modules is desirable, yet is currently not readily accomplished.